1. Field of the Invention
The present disclosure relates to an improved method of producing terminally differentiated neuronal cells such as GABAergic neurons from pluripotent stem cells such as murine embryonic stem cells or human embryonic stem cells. The GABAergic neurons generated according to the present disclosure may serve as an excellent source for cell replacement therapy in neurodegenerative disorders and neuronal diseases such as, for example, stroke, ischemia, Parkinson's disease, Alzheimer's disease, epilepsy, and Huntington's disease.
2. Description of Related Art
Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system (CNS), and is widely distributed throughout the brain and expressed in interneurons modulating local circuits. GABAergic neurons, which produce GABA, are the predominant inhibitory neurons in the mammalian CNS, and approximately 60-75% of all synapses in the CNS are GABAergic (Schwartz, R. D., 1988, Biochem. Pharmacol. 37:3369-75). GABAergic neurons are localized in the hippocampus, cerebellum, cerebral cortex, and hypothalamus, and GABA binds to at least three receptors, including GABA-A and GABA-B. GABA-A receptors mediate fast inhibitory synaptic transmissions, neuronal excitability, and rapid changes in mood, such as seizure threshhold, anxiety, panic, and response to stress (i.e., the “fight or flight” response). GABA-A receptors are also binding sites for benzodiazepines, ethanol, barbiturates, and neurosteroids. GABA-B receptors mediate slow inhibitory transmissions, and may be important in memory, mood, and pain.
The pathogenesis of several neurological disorders appears to involve a decrease in GABAergic neurotransmission, including some forms of epilepsy, chronic pain, anxiety, and other mood disorders. For example, a positron emission tomography (PET) study showed that patients with panic disorder have decreased GABA-A receptor binding (Malizia et al., 1998, Arch. Gen. Psychiatry 55:715-20). In addition, low plasma GABA may be characteristic of a subgroup of patients with mood disorders (Brambilla et al., 2003, Mol. Psychiatry 8:721-37). Certain drugs that enhance GABA activity have been shown effective in the treatment of these disorders, such as benodiazepines, valproate, and phenobarbital.
Many diseases of the central nervous system (CNS) such as Parkinson's disease, Alzheimer's disease, Multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, cerebral ischemia, and stroke are characterized by degeneration of neurons in the brain and spinal cord regions. Cells or neurons that degenerate or are otherwise damaged are not intrinsically replaced or repaired by the body, which can lead to permanent and irreversible damage (During et al., 2001, Human Gene Ther. 12:1589-1591). Stroke or cerebral ischemia can occur when a blood clot blocks a blood vessel or artery, interrupting blood flow to an area of the brain, which causes the death of brain cells in the immediate area of the block. The brain cells in the infarct usually die within minutes to a few hours after the stroke or ischemia occurs. The death of these cells can lead to a release of chemicals that set off a chain reaction called the “ischemic cascade,” which endangers brain cells in the larger, surrounding area of brain tissue for which the blood supply is compromised. Without prompt medical treatment, this larger area of brain cells may also die, which can cause even more severe and long-term damage to the brain. Given the rapid pace of the ischemic cascade, the “window of opportunity” for interventional treatment is only about six hours. Beyond this window of time, reestablishment of blood flow and administration of neuroprotective agents may fail to help and can potentially cause further damage to brain functions (Padosch et al., 2001, Anaesthesist 50:905-920; Nishino and Borlongan, 2000, Prog. Brain Res. 127:461-476).
When stroke occurs, the disruption of blood flow to the brain has a detrimental and potentially fatal effect on individual or groups of neurons. Starving large numbers of neurons of oxygen and vital nutrients in a specific area of the brain due to cerebral ischemia can lead to severe loss of functional capabilities in patients. For example, stroke patients may experience loss of speech, memory, cognition, reduced mobility, or even paralysis. Without an adequate blood supply, brain cells lose their ability to produce energy, particularly adenosine triphosphate (ATP). If critical thresholds of this energy failure occur, brain cells are damaged and die. Many researchers believe that an immense number of mechanisms cause brain cell damage and death following energy failure, with each of these mechanisms representing a potential route for therapeutic intervention.
One way brain cells respond to energy failure is by elevating the concentration of intracellular calcium. These concentrations can be driven to dangerous levels by a process called excitotoxicity, in which brain cells release excessive amounts of glutamate, a neurotransmitter, which leads to the degradation and destruction of vital cells located in the hippocampus, cortex, and thalamus region of the brain (Nishino and Borlongan, 2000, Prog. Brain Res. 127:461-476). In addition, GABA-producing cells in the hippocampus region of the brain often degenerate after a stroke (Nishino and Borlongan, 2000, Prog. Brain Res. 127:461-476). Based on neurohistopathological and neuropsychological investigations, several neuroprotective drug therapies have been developed to treat neurological disorders associated with cerebral ischemia or stroke, such as GABAergic agonists, calcium antagonists, glutamate antagonists, and antioxidants (Stutzmann et al., 2002, CNS Drug Rev. 8:1-30; Rochelle et al., 2001, J. Neurochem. 77:353-371; Blezer et al., 2002 Eur. J. Pharmacol. 444:75-81). Currently there are hundreds of drugs and compounds in various stages of development for the prevention and acute interventional treatment of stroke (Rochelle et al., 2001, J. Neurochem. 77:353-371). It is anticipated that several of these drugs will be submitted to the FDA for approval, and many are already engaged in the last phase of clinical trials. Among these, GABAergic drugs are found to be exceptionally effective in treating neurological disorders associated with cerebral ischemia or stroke.
Given the multi-dimensional nature of ischemic brain cell injury, however, stroke experts predict that no single drug-based therapy will be able to completely protect the brain during and after a stroke. Since current therapeutic alternatives do not adequately treat damage associated with cerebral ischemia or stroke, there is great interest in developing alternative therapies for various neurodegenerative disorders and neuronal diseases. A cell-based therapy may be the only means available for comprehensively treating the damage caused by such an event. Many neurological diseases and conditions are caused by the loss of neuronal cells in the brain and spinal cord regions. A wide spectrum of these neurological diseases and conditions, including but not limited to Parkinson's disease, Alzheimer's disease, Huntington's disease, and spinal cord injury, may be treatable with cell based therapies. For example, patients with Parkinson's disease have been successfully treated by transplanting dopaminergic neurons into the brain of affected individuals (Grisolia, 2002, Brain Res Bull 57:823-826). Therefore, when GABA-producing cells are affected or damaged in cerebral ischemia or stroke patients, the replacement of these damaged GABA-producing cells with new and healthy GABA-producing cells would be an ideal therapy for the treatment of cerebral ischemia or stroke.
One major problem with cell transplantation as a therapeutic option for neurodegenerative disorders and neuronal diseases is the need for large quantities of neuronal cells, which are difficult to isolate from fetal or adult sources. One solution to this dilemma is the availability of pluripotent stem cells, which can be used to generate unlimited numbers of terminally differentiated cell types. Pluripotent embryonic stem (ES) cells are a viable alternative source of neuronal cells that may be used to treat various neurodegenerative disorders and neuronal diseases. ES cells can proliferate indefinitely in an undifferentiated state and are pluripotent, which means they are capable of differentiating into nearly all cell types present in the body. Because ES cells are capable of becoming almost all of the specialized cells of the body, they have the potential to generate replacement cells for a broad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilage, and the like. ES cells can be derived from the inner cell mass (ICM) of a blastocyst, which is a stage of embryo development that occurs prior to implantation. Human ES cells may be derived from a human blastocyst at an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. ES cells derived from the ICM can be cultured in vitro and under the appropriate conditions proliferate indefinitely.
ES cell lines have been successfully established for a number of species, including mouse (Evans et al., 1981, Nature 292:154-156), rat (Iannaccone et al., 1994, Dev. Biol., 163:288-292), porcine (Evans et al., 1990, Theriogenology 33:125-128; Notarianni et al., 1990, J. Reprod. Fertil. Suppl. 41:51-6), sheep and goat (Meinecke-Tillmann and Meinecke, 1996, J. Animal Breeding and Genetics 113:413-426; Notarianni et al., 1991, J. Reprod. Fertil. Suppl. 43:255-60), rabbit (Giles et al., 1993, Mol. Reprod. Dev. 36:130-138; Graves et al., 1993, Mol. Reprod. Dev. 36:424-433), mink (Sukoyan et al., Mol. Reprod. Dev. 1992, 33:418-431), hamster (Doetschman et al., 1988, Dev. Biol. 127:224-227), domestic fowl (Pain et al., 1996, Development 122(8):2339-48), primate (U.S. Pat. No. 5,843,780), and human (Thomson et al., 1998, Science 282:1145-1147; Reubinoff et al., 2000, Nature Biotech. 18:399-403). Like other mammalian ES cells, human ES cells differentiate and form tissues of all three germ layers when injected into immunodeficient mice, proving their pluripotency. Published reports show that human ES cells have been maintained in culture for more than a year during which time they retained their pluripotency, self-renewing capacity, and normal karyotype (Thomson et. al., 1995, PNAS 92:7844-7848).
ES cells have been shown to differentiate into neurons and glial cells in both in vitro models (Bain et al., 1995, Dev. Biol. 168:342-357), as well as in vivo models (Brustle, et al., 1999, Science 285:754-56). Similarly, blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation (Deacon et al., 1998, Exp. Neurol. 149:28-41). Human or rodent stems cells are able to differentiate into specific neuronal types when grafted into either a developing central nervous system (Flax et al., 1998, Nat. Biotechnol. 16:1033-39; Brustle et al., 1998, Nat. Biotechnol. 16:1040-44; Reubinoff et al., 2001, Nat. Biotechnol. 19:1034-40) or neurogenic areas of the adult CNS (Fricker et al., 1999, J. Neurosci. 19:5990-6005; Shihabuddin et al., 2000, J. Neurosci. 20:8727-35).
One method for generating GABA-producing cells from immature neuronal cells has been reported (Rubenstein et al., U.S. Pat. No. 6,602,680, incorporated herein by reference). Rubenstein et al. reported the production of GABAergic cells by increasing the activity of a DLX gene, for example DLX1, DLX2, or DLX5, in an immature neuronal cells. The increase in DLX activity causes differentiation of the immature neuronal cells into cells with the GABAergic phenotype. Methods for deriving GABA-producing cells from mouse embryonic stem cells have also been reported (Hancock et al., 2000, Biochem. Biophys. Res. Commun. 271(2):418-21, Westmoreland et al., 2001, Biochem. Biophys. Res. Commun. 284(3):674-80; U.S. Publication No. 2003/0036195 A1, each specifically incorporated herein by reference), but these methods do not generate high percentages of GABAergic neurons. Since large numbers of GABAergic neurons are required for cell replacement therapy, there is a need for additional in vitro methods for generating large numbers of GABAergic neurons from pluripotent stem cells.
Methods that can generate high yields of GABAergic neurons have great clinical significance for cell transplantation therapy, particularly for patients suffering from cerebral ischemia or stroke. To date, available therapies are extremely limited for treating the neuropathology associated with cerebral ischemia and stroke, so there is great interest in developing alternative therapies. Cell-based therapies will require large numbers of cells or neurons for treatment, which is not possible if fetal or adult tissue is the only source available for the cells and neurons. For example, about 1 million dopaminergic producing cells must be transplanted into a single Parkinson's disease animal model to study the functional recovery of motor function (Grisolia, 2002, Brain Res. Bull. 57:823-826). Obtaining such a large number of cells using fetal material raises many ethical problems. Generation of GABAergic neurons from pluripotent stem cells offers a potentially unlimited supply of GABAergic neurons to use in cell based-therapies. But methods that yield high percentages of GABAergic neurons are necessary to make this source practical, particularly since it is likely that large numbers of GABAergic neurons will be needed for therapeutic methods utilizing these cells.